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Characteristics of Marrow Production and Reticulocyte Maturation in Normal Man in Response to Anemia

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Characteristics of Marrow Production and Reticulocyte Maturation in Normal Man in Response to Anemia Characteristics of marrow production and reticulocyte maturation in normal man in response to anemia Robert S. Hillman J Clin Invest. 1969;48(3):443-453. https://doi.org/10.1172/JCI106001. Research Article Erythropoiesis in normal man was studied during periods of phlebotomy-induced anemia of varying severity. This study permitted a comparison of marrow production measurements over a wide range of marrow production levels. As long as the serum iron remained above 50 μg/100 ml, measurements of plasma iron turnover provided an excellent index of marrow production at all levels of red cell production. In contrast, the absolute reticulocyte count demonstrated a poor correlation with the other measurements. This was shown to be the result of a prolongation of the time required for circulating reticulocytes to lose their reticulum, which correlated with the severity of the anemia. For the clinical application of the reticulocyte count as a measurement of marrow production, an adjustment must be made for this alteration in the circulating reticulocyte maturation time. Find the latest version: https://jci.me/106001/pdf Characteristics of Marrow Production and Reticulocyte Maturation in Normal Man in Response to Anemia ROBERT S. HILLMAN From the Department of Medicine, University of Washington School of Medicine, Seattle, Washington 98104 A B S T R A C T Erythropoiesis in normal man was stud- normal human erythroid marrow to graded levels of ied during periods of phlebotomy-induced anemia of anemia maintained for 3-5 wk. This examination per- varying severity. This study permitted a comparison of mitted a comparison of erythropoietic measurements marrow production measurements over a wide range of over a wide range of marrow production levels and a marrow production levels. As long as the serum iron definition of the changes in reticulocyte maturation remained above 50 ug/100 ml, measurements of plasma which are important to the interpretation of the reticu- iron turnover provided an excellent index of marrow locyte count as a production measurement. production at all levels of red cell production. In con- trast, the absolute reticulocyte count demonstrated a METHODS poor correlation with the other measurements. This was All studies were performed on the Clinical Research Center shown to be the result of a prolongation of the time re- of the King County Hospital, Seattle, Washington. Partici- quired for circulating reticulocytes to lose their reticu- pants included two patients with hemochromatosis, who re- lum, which correlated with the severity of the anemia. quired prolonged phlebotomy threapy, and six normal volun- For the clinical application of the reticulocyte count as teers. The subjects were males between the ages of 23 and 48. All studies were carried out according to the guidelines of a measurement of marrow production, an adjustment the Helsinki Declaration for participation of human volun- must be made for this alteration in the circulating retic- teers. Before study, each individual was carefully evaluated ulocyte maturation time. to exclude renal impairment or other significant illnesses which could have interfered with maximum erythropoietin INTRODUCTION output and marrow response to phlebotomy. During an ini- tial 10 day observation, base line studies of marrow func- Erythropoiesis in man has been studied by a number of tion were obtained, including two measurements of plasma techniques, including: the reticulocyte count, plasma iron iron turnover, a marrow iron transit time, serum iron, and turnover, 51Cr or diisopropylfluorophosphate (DF"P) total iron binding-capacity determinations, a 'Cr-labeled red red cell life-span, anid stercobilinogen excretion. Except cell mass, mean cell indices, repeated reticulocyte counts, and a bone marrow examination with Prussian blue stain for the reticulocyte count, these measurements are in for iron stores. Throughout the control and subsequent study essential agreement and apparently correlate with the periods, the subjects were kept on a high protein diet, and actual level of erythroid marrow production. However, folic acid was provided in excess, 5 mg twice a day orally. this conclusion is based primarily on measurements in In order to investigate the role of iron in the marrow pro- duction response, iron was provided by varying methods pathological states (1-5). Studies of the normal marrow when required (13). have been restricted to characterization of basal produc- After completion of base line studies, the subjects were tion and the acute response of marrow to sudden anoxia, phlebotomized, letting 500-1000 ml of whole blood to lower phlebotomy, or transfusion polycythemia (1-3, 6-10). the hematocrit to a level of 32-37%. The total blood volume As for the reticulocyte count, it has generally been dis- was kept constant by immediate infusion of an equal quan- tity of 5% albumin solution. Phlebotomies of more than 500 carded because of a poor correlation with the other pro- ml were accomplished in two stages by complete replace- duction measurements (11, 12). ment of the plasma volume after removal of each 500 ml. The present study has examined the response of the Over the next 3-5 wk, the hematocrit was maintained at this level by graded daily phlebotomy adjusted to remove Received for publication 20 June 1968 and in revised form enough red cells to compensate for increased production. 4 November 1968. An equal volume of 5%o albumin solution was infused with The Journal of Clinical Investigation Volume 48 1969 443 any phlebotomy of more than 50 ml of whole blood.' The to 0-4°C, centrifuged, and the separated plasma was counted erythroid marrow production response was then monitored in a gamma-well counter2 to 10,000 or more counts above with twice daily reticulocyte counts, measurements of the background for ± S1% accuracy. The plasma iron turnover plasma iron turnover at 7-day intervals, and from the level was calculated according to Bothwell and Finch (14) with of daily phlebotomy required to maintain the hematocrit at the mean of two serum irons drawn at 0 time and 30 min this level (see below, phlebotomy production measurement). after radioiron injection and the tj clearance. The radioiron With the hematocrit maintained at 32-37%, the erythroid administered to any single patient was limited to a maxi- marrow production response was measured in eight indi- mum of 25 uc for the entire study. viduals, six normals relying on either reticuloendothelial The plasma iron turnover production index was calculated cell iron stores and (or) orally administered iron, and two according to Giblett et al. (1) with 0.65 mg/100 ml of hemochromatosis patients with excessive parenchymal and whole blood per 24 hr as normal. reticuloendothelial cell iron stores. Patients on orally ad- Patient's PIT ministered iron received a 300 mg ferrous gluconate tablet PIT production index = -a0 65 at 2-hr intervals while awake for a total of 8-9 doses/day. Marrow production responses were monitored until produc- In order to study the effect of varying levels of reticulo- tion had plateaued for at least 10 days. This plateau was cytosis on the plasma iron turnover measurement, direct considered to represent a maximum level of marrow produc- reticulocyte uptake of radioiron was measured with each tion for the selected conditions of anemic stress and iron plasma iron turnover by in vitro incubation of the patient's supply. blood with '9Fe-labeled transferrin. Whole blood was col- Subsequently, six of the normal volunteers and one sub- lected in heparin, centrifuged, and the plasma removed. ject with hemochromatosis were studied at a hematocrit After adjusting the plasma iron to 200-250 ,tg/100 ml with level of 25-30%, maintained over a 3-5 wk period. This ferrous citrate-59Fe, sufficient plasma was recombined with level of phlebotomy-induced anemic stress was tolerated by the red cell fraction to obtain a hematocrit of 50-60%. the subjects without difficulty. One individual worked full Once a zero time sample was obtained the red cell suspen- time cleaning and steaming Alaskan King crab; the others sion was incubated in a water bath at 37°C. Samples were continued their normal daily activities without restriction. removed at 60 and 120 min, immediately chilled to 0-40C, centrifuged, and plasma aliquots obtained for counting. The Marrow production studies red cell fraction was washed three times in cold saline, Plasma iron turnover measurements. All determinations lysed in distilled water, and an aliquot counted for radio- of the plasma iron turnover were performed at 10:00 a.m. in activity. Red cell iron uptake was calculated from the se- a fasting state. With patients on orally administered iron rum iron and the net gain in activity of the red cell frac- the radioiron was injected 1 hour after the last oral dose. tion over time zero. 1-10 uc of ferrous citrate-"Fe was incubated with 5-10 ml Phlebotomy production measurements. Production was of the patient's plasma for 30 min before injection. The in- also estimated from the level of daily phlebotomy, because jected dose and standard dilution were determined gravi- if the hematocrit is kept at a constant level, red cell produc- metrically. Accurately timed venous samples were obtained tion must equal the amount of packed red cells removed at frequent intervals over the next 2 hr, immediately chilled by phlebotomy plus the amount of red cells dying each day. 1 5%o albumin solution, Albumisol (Merck, Sharp & Dohme, West Point, Pa.) was administered in equal volumes for any phlebotomy exceeding 50 ml of whole blood. Repeated blood volumes (51Cr-labeled red cell mass and (or) radioiron-labeled transferrin plasma volume) at weekly intervals confirmed the fact that this technique kept the blood volume constant throughout the periods of prolonged phlebotomy. Hct 32-377( Hct 25-30% Week ..... I 2 3 4 5 6 7 8 9 Patient 1 5150 5200 5400 5210 5050 5320 5180 5060 2 3780 3800 3655 - 3850 4050 3780 3820 3650 3 4320 4550 4450 4300 4610 4400 - 4510 4480 'Nuclear-Chicago gamma-well counter, Nuclear-Chicago Corporation, Des Plaines, Ill.
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